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Combination of mTOR and EGFR kinase inhibitors blocks mTORC1 and mTORC2 kinase activity and suppresses the progression of colorectal carcinoma.

Wang Q, Wei F, Li C, Lv G, Wang G, Liu T, Bellail AC, Hao C - PLoS ONE (2013)

Bottom Line: To test this notion, we showed that the combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity.In addition, we showed that the combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death.A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastrointestinal Surgery, First Hospital of Jilin University, Changchun, Jilin, China. wangquan-jlcc@hotmail.com

ABSTRACT
Mammalian target of rapamycin complex 1 and 2 (mTORC1/2) are overactive in colorectal carcinomas; however, the first generation of mTOR inhibitors such as rapamycin have failed to show clinical benefits in treating colorectal carcinoma in part due to their effects only on mTORC1. The second generation of mTOR inhibitors such as PP242 targets mTOR kinase; thus, they are capable of inhibiting both mTORC1 and mTORC2. To examine the therapeutic potential of the mTOR kinase inhibitors, we treated a panel of colorectal carcinoma cell lines with PP242. Western blotting showed that the PP242 inhibition of mTORC2-mediated AKT phosphorylation at Ser 473 (AKT(S473)) was transient only in the first few hours of the PP242 treatment. Receptor tyrosine kinase arrays further revealed that PP242 treatment increased the phosphorylated epidermal growth factor receptor (EGFR) at Tyr 1068 (EGFR(T1068)). The parallel increase of AKT(S473) and EGFR(T1068) in the cells following PP242 treatment raised the possibility that EGFR phosphorylation might contribute to the PP242 incomplete inhibition of mTORC2. To test this notion, we showed that the combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity. In addition, we showed that the combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death. A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice. This study suggests that the combination of mTOR kinase and EGFR inhibitors may provide an effective treatment of colorectal carcinoma.

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PP242 treatment leads to the EGFR phosphorylation in the carcinoma cells.(A) RTK arrays of DLD-1 cells untreated or treated with rapamycin (0.1 μM) and PP242 (1 μM) for 72 hours show the phosphorylated EGFR (p-EGFR) as indicated by arrows. (B) DLD-1 and HT29 cells grown in EGF (50 ng/ml) containing medium were treated with rapamycin (0.1 μM), PP242 (1 μM), or LY294002 (20 μM) for the indicated hours and examined by western blotting for the presence of the phosphorylated and unphosphorylated proteins (left). (C) DLD-1 and HT29 cells grown in EGF (50 ng/ml) containing medium were treated with PP242 (1 μM) or BKM120 (1 μM) for the indicated hours and tested by western blotting for the presence of the proteins (left).
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pone-0073175-g002: PP242 treatment leads to the EGFR phosphorylation in the carcinoma cells.(A) RTK arrays of DLD-1 cells untreated or treated with rapamycin (0.1 μM) and PP242 (1 μM) for 72 hours show the phosphorylated EGFR (p-EGFR) as indicated by arrows. (B) DLD-1 and HT29 cells grown in EGF (50 ng/ml) containing medium were treated with rapamycin (0.1 μM), PP242 (1 μM), or LY294002 (20 μM) for the indicated hours and examined by western blotting for the presence of the phosphorylated and unphosphorylated proteins (left). (C) DLD-1 and HT29 cells grown in EGF (50 ng/ml) containing medium were treated with PP242 (1 μM) or BKM120 (1 μM) for the indicated hours and tested by western blotting for the presence of the proteins (left).

Mentions: RTKs activates mTOR kinase through PI3K-AKT pathway [51]; thus to examine whether PP242 treatment may activate RTKs, we treated DLD-1 cells either with rapamycin (0.1 μM) or PP242 (1 μM) for 72 hours and examined the cell lysates for RTK phosphorylation using a RTK phosphorylation array. Interestingly, the array detected phosphorylated EGFR in the cells treated with PP242, but not treated with rapamycin and untreated (Figure 2A). To further examine whether and how rapamycin and PP242 affect the EGFR pathway in the carcinoma cells, the colorectal carcinoma cell lines DLD-1 and HT29 were grown in 1% FBS culture medium containing 50 ng/ml of EGF. Since this experimental design mimics the endogenous EGF-EGFR pathway in the cancer, it is often used in study of the pathway in cancer cells. The cells were treated either with rapamycin or PP242; western blotting showed that the treatment of rapamycin did not affect the levels of either unphosphorylated or phosphorylated EGFR at T1068 (EGFRT1068) in the cell lines (Figure 2B); the data indicate that rapamycin did not activate EGFR. Detection of phosphorylated AKT (AKTS473) kept in line with the report that rapamycin does not inhibit mTORC2 [22].


Combination of mTOR and EGFR kinase inhibitors blocks mTORC1 and mTORC2 kinase activity and suppresses the progression of colorectal carcinoma.

Wang Q, Wei F, Li C, Lv G, Wang G, Liu T, Bellail AC, Hao C - PLoS ONE (2013)

PP242 treatment leads to the EGFR phosphorylation in the carcinoma cells.(A) RTK arrays of DLD-1 cells untreated or treated with rapamycin (0.1 μM) and PP242 (1 μM) for 72 hours show the phosphorylated EGFR (p-EGFR) as indicated by arrows. (B) DLD-1 and HT29 cells grown in EGF (50 ng/ml) containing medium were treated with rapamycin (0.1 μM), PP242 (1 μM), or LY294002 (20 μM) for the indicated hours and examined by western blotting for the presence of the phosphorylated and unphosphorylated proteins (left). (C) DLD-1 and HT29 cells grown in EGF (50 ng/ml) containing medium were treated with PP242 (1 μM) or BKM120 (1 μM) for the indicated hours and tested by western blotting for the presence of the proteins (left).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3750018&req=5

pone-0073175-g002: PP242 treatment leads to the EGFR phosphorylation in the carcinoma cells.(A) RTK arrays of DLD-1 cells untreated or treated with rapamycin (0.1 μM) and PP242 (1 μM) for 72 hours show the phosphorylated EGFR (p-EGFR) as indicated by arrows. (B) DLD-1 and HT29 cells grown in EGF (50 ng/ml) containing medium were treated with rapamycin (0.1 μM), PP242 (1 μM), or LY294002 (20 μM) for the indicated hours and examined by western blotting for the presence of the phosphorylated and unphosphorylated proteins (left). (C) DLD-1 and HT29 cells grown in EGF (50 ng/ml) containing medium were treated with PP242 (1 μM) or BKM120 (1 μM) for the indicated hours and tested by western blotting for the presence of the proteins (left).
Mentions: RTKs activates mTOR kinase through PI3K-AKT pathway [51]; thus to examine whether PP242 treatment may activate RTKs, we treated DLD-1 cells either with rapamycin (0.1 μM) or PP242 (1 μM) for 72 hours and examined the cell lysates for RTK phosphorylation using a RTK phosphorylation array. Interestingly, the array detected phosphorylated EGFR in the cells treated with PP242, but not treated with rapamycin and untreated (Figure 2A). To further examine whether and how rapamycin and PP242 affect the EGFR pathway in the carcinoma cells, the colorectal carcinoma cell lines DLD-1 and HT29 were grown in 1% FBS culture medium containing 50 ng/ml of EGF. Since this experimental design mimics the endogenous EGF-EGFR pathway in the cancer, it is often used in study of the pathway in cancer cells. The cells were treated either with rapamycin or PP242; western blotting showed that the treatment of rapamycin did not affect the levels of either unphosphorylated or phosphorylated EGFR at T1068 (EGFRT1068) in the cell lines (Figure 2B); the data indicate that rapamycin did not activate EGFR. Detection of phosphorylated AKT (AKTS473) kept in line with the report that rapamycin does not inhibit mTORC2 [22].

Bottom Line: To test this notion, we showed that the combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity.In addition, we showed that the combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death.A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Gastrointestinal Surgery, First Hospital of Jilin University, Changchun, Jilin, China. wangquan-jlcc@hotmail.com

ABSTRACT
Mammalian target of rapamycin complex 1 and 2 (mTORC1/2) are overactive in colorectal carcinomas; however, the first generation of mTOR inhibitors such as rapamycin have failed to show clinical benefits in treating colorectal carcinoma in part due to their effects only on mTORC1. The second generation of mTOR inhibitors such as PP242 targets mTOR kinase; thus, they are capable of inhibiting both mTORC1 and mTORC2. To examine the therapeutic potential of the mTOR kinase inhibitors, we treated a panel of colorectal carcinoma cell lines with PP242. Western blotting showed that the PP242 inhibition of mTORC2-mediated AKT phosphorylation at Ser 473 (AKT(S473)) was transient only in the first few hours of the PP242 treatment. Receptor tyrosine kinase arrays further revealed that PP242 treatment increased the phosphorylated epidermal growth factor receptor (EGFR) at Tyr 1068 (EGFR(T1068)). The parallel increase of AKT(S473) and EGFR(T1068) in the cells following PP242 treatment raised the possibility that EGFR phosphorylation might contribute to the PP242 incomplete inhibition of mTORC2. To test this notion, we showed that the combination of PP242 with erlotinib, an EGFR small molecule inhibitor, blocked both mTORC1 and mTORC2 kinase activity. In addition, we showed that the combination treatment inhibited colony formation, blocked cell growth and induced apoptotic cell death. A systemic administration of PP242 and erlotinib resulted in the progression suppression of colorectal carcinoma xenografts in mice. This study suggests that the combination of mTOR kinase and EGFR inhibitors may provide an effective treatment of colorectal carcinoma.

Show MeSH
Related in: MedlinePlus